The majority of recombinant therapeutic proteins display one or more post-translational modifications. These modifications may occur during their ribosomal synthesis or (more usually) after synthesis is complete. A large number of post-translational modifications have been characterized to date, and these modifications may impart some structural aspect or functional role of the affected protein. Common post-translational modifications associated with therapeutic proteins include carboxylation and hydroxylation, amidation and sulfation, disulfide bond formation and proteolytic processing, as well as glycosylation, isomerization, oxidation, cyclization of a N-terminal glutamine or glutamic acid to pyro-glutamic acid, fragmentation, and aggregation. Thus, post-translational modifications may either be caused by enzymatic modification of the protein or by non-enzymatic conversion and post-translational modifications may occur inside the expression host, during cell culture, or during purification or storage of the protein.
Recombinant monoclonal antibodies are becoming of great value for the biotechnology industry and numerous antibodies have been approved for treating a variety of diseases. As antibodies generally are produced in mammalian cells, such as CHO cells, they may have a number of different post-translational modifications, which lead to heterogeneity in the product. Heterogeneity may be caused by changes in the surface charge of the antibody, either directly, as a change in the number of charged residues, or indirectly as a chemical or physical alteration that changes surface-charge distribution such as glycosylation, carboxypeptidase clipping of the C-terminal lysine of the heavy chain, and cyclization of a N-terminal glutamine or glutamic acid residue to pyro-glutamic acid.
Antibodies are typically made of basic structural units, each with two large heavy chains and two small light chains joined via disulfide bridges and non-covalent interactions. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. For example there are four isotypes of human IgG (1 through 4), depending on the gene for the heavy chain constant region. The light chain constant domain is coded by two genes (κ or λ). Consequently, each IgG isotype can be either κ or λ, for instance IgG1κ. Although there are several types of antibodies produced in different types of cell lines the most clinically significant antibodies are full-length antibodies of the IgG1 or IgG2 types.
Many of the human IgG1 or IgG2 types antibodies contain a glutamic acid (Glu) and/or a glutamine (Gln) residue at the N-terminus of either the light chain or the heavy chain or both. A significant portion of light chain genes codes for either glutamic acid or glutamine. Such N-terminal glutamic acid and/or glutamine residues may undergo cyclization to form pyro-glutamate (pGlu) as shown in FIG. 1. Pyro-glutamate formation therefore occurs in virtually all clinically significant antibodies and different levels of completeness of the process are a common source of heterogeneity. This heterogeneity is not desired in therapeutic antibodies, since these changes can alter surface charge properties of the antibody either directly by changing the number of charged groups or indirectly by introducing structural alterations. Such modifications have the potential to decrease biological activity, as well as alter pharmacokinetics and antigenicity.
During cyclization of glutamine, the N-terminal primary amine (positively charged at a neutral pH) is converted to a neutral amide, resulting in a change of the net charge of the antibody. The reaction is accompanied by a loss of ammonia (17 Da). Consequently, the lack of cyclization may be detected as basic variants by cation exchange chromatography, since the main peak is typical the fully cyclized species or as late-eluting peaks by reversed-phase HPLC due to the increased hydrophobicity after the loss of the N-terminal amine.
Cyclization of glutamic acid occurs via the carboxyl group of the side chain and the N-terminal amine, thus forming a neutral amide and releasing water (18 Da). The net charge remains the same because one acidic and one basic group condense in the reaction, however, the loss of two charged residues increases the hydrophobicity of the molecule, allowing detection by reversed-phase HPLC.
The mechanism of pyro-glutamic acid formation in antibodies is not fully understood. Cyclization can occur spontaneously or it can be aided by an enzyme glutaminyl cyclase. It remains unclear whether glutaminyl cyclase is active in the CHO cell line, which is most commonly used for antibody production; however, rates of spontaneous cyclization indicate that the reaction is likely non-enzymatic.
For instance Yu et al. (Journal of Pharmaceutical and Biomedical Analysis 2006; 42:455-463) investigated the non-enzymatic pyro-glutamate formation from glutamic acid (Glu or E) at the N-terminus of both the light chain and the heavy chain of a monoclonal antibody for a period of 3 months. Yu et al. states that this non-enzymatic cyclization of Glu to pGlu of mAbs could be one of the major degradation pathways incurred in the mAb production and storage depending on pH and temperature conditions during the process development. They concluded that whether such pyro-Glu may induce further modifications and alter the mAb bioactivity or therapeutic potency is unclear, and they proposed to closely monitoring N-terminal pGlu formation since it can be critical to ensure quality of mAb therapeutics with N-terminal Glu.
Chelius et al. (Anal. Chem. 2006, 78, 2370-2376) also investigated the non-enzymatic pyro-glutamate formation from glutamic acid at the N-terminus of both the light chain and the heavy chain of several monoclonal antibodies and found that this non-enzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37° C. and 45° C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyro-glutamic acid at pH 6.2 and increased formation of pyro-glutamic acid at pH 4 and pH 8.
Having regard to the conversion of glutamine (Gln or Q) to pyro-glutamate Dick et al. (Biotechnology and Bioengineering, Vol. 97, No. 3, Jun. 15, 2007) showed that such cyclization of the N-terminal glutamine of a recombinant monoclonal antibody occurs spontaneously and concluded that the near complete conversion observed in many final container monoclonal antibodies is most likely caused by a combination of bioreactor incubation and purification conditions with a majority of the modification occurring in the bioreactor. This study proves that the commonly observed pyro-Q variant in many recombinant antibodies is caused inside the bioreactor with only a small contribution from the purification process, and is accelerated by high temperature and solvent composition. Specifically, the higher conversion is found at 37° C. and in a 35 mM phosphate buffer with 75 mM NaCl (pH 6.2).
Thus, post-translational modifications such as cyclization of N-terminal glutamine or glutamic acid residue to pyro-glutamic acid lead to heterogeneity of the expressed protein that may differ from batch to batch due to slight variations in production and purification conditions. Therefore, one of the more difficult challenges for producing a biosimilar protein is to match the heterogeneity of the innovator product. So, a more robust and reproducible industrial large-scale purification process with analytical support is needed to satisfy the stringent purity requirements for pharmaceutical proteins, such as antibodies.